1. Field of the Invention
The present invention relates generally to the field of treatment of autoimmune disease, such as multiple sclerosis (MS). More particularly, it concerns a T-cell receptor sequence found in some MS patients, and methods for its detection.
2. Description of Related Art
In humans and other mammals, T cell receptors are found on T cells. T cell receptors comprise xcex1 and xcex2 chains, with xcex2 chains comprising the following regions from N-terminus to C-terminus: Vxcex2-Dxcex2-Jxcex2-Cxcex2. T cell receptors naturally vary in the Vxcex2-Dxcex2-Jxcex2 regions.
When an antigen is presented to the T cells by an antigen-presenting cell (APC), a T cell receptor with variable regions (including Vxcex2-Dxcex2-Jxcex2) that so happen to recognize the antigen binds to the antigen on the APC. The T cell bearing the T cell receptor then undergoes activation (clonal expansion).
The pathogenesis of a number of autoimmune diseases is believed to lie in autoimmune T cell responses to antigens presented normally by the organism. An example of such a disease is multiple sclerosis (MS), which is generally held to arise in T cell responses to myelin antigens, in particular myelin basic protein (MBP). MBP-reactive T cells are found to undergo in vivo activation, and occur at a higher precursor frequency in blood and cerebrospinal fluid in patients with MS as opposed to control individuals. These MBP-reactive T cells produce Th1 cytokines, e.g. IL-2, TNF, and xcex3-interferon. These Th1 cytokines facilitate migration of inflammatory cells into the central nervous system and exacerbate myelin-destructive inflammatory responses in MS.
A number of regulatory mechanisms can be made use of in the treatment of MS. One such is vaccination with one or more of the limited number of T cell membrane-associated peptides with extracellular domains. Vandenbark, U.S. Pat. No. 5,614,192, discloses treatment of autoimmune diseases by the use of immunogenic T cell receptor peptides of 15 to 30 amino acids comprising at least part of the second complementarity determining region (CDR2) of the T cell receptor. A copending U.S. Patent Application by Zhang (60/099,102) discloses treatment of autoimmune diseases by use of immunogenic T cell receptor peptides in combination with immunogenic T cell activation marker peptides.
One area in which vaccination with T cell receptor peptides can be improved is by determining which, if any, common motifs are found in the T cell receptors of a patient with an autoimmune disease such as MS. If such motifs are found, then the patient can be vaccinated with peptides identical to the motifs, in order to facilitate treatment.
Therefore, it is desirable to have the amino acid sequences of common motifs found in the T cell receptors of patients with autoimmune diseases. It is also desirable to be able to readily detect such motifs in a patient sample by a convenient method, such as PCR. In addition, it is desirable to use peptides identical to the detected motifs to treat a patient with the autoimmune disease.
The present invention discloses such a common motif found in the T cell receptors of a subset of Vxcex213.1 T cells, the xe2x80x9cLGRAGLTY motifxe2x80x9d, which has the amino acid sequence Lue Gly Arg Ala Gly Leu Thr Tyr (SEQ ID NO: 3), as well as a method for its ready detection by PCR. This motif is found in some T cell receptors of some T cells that recognize amino acids 83-99 of MBP (hereinafter xe2x80x9cMBP83-99xe2x80x9d). The motif in the context of this subset of Vxcex213.1 T cells may hereinafter be referred to as xe2x80x9cVxcex213.1-LGRAGLTY.xe2x80x9d Peptides identical to the motif can be used to vaccinate patients in order to treat or prevent autoimmune diseases with which Vxcex213.1-LGRAGLTY is associated. One such autoimmune disease is MS.
In one embodiment, the present invention is directed to an oligonucleotide from about 15 to 30 nucleotides in length which comprises at least 10 contiguous nucleotides of SEQ ID NO:1, or a sequence complementary thereto or derived therefrom. Even more preferable is an oligonucleotide, of about 15 to 30 nucleotides in length, which comprises 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of SEQ ID NO:, or a sequence complementary thereto. Most preferable is an oligonucleotide, of about 15 to 30 nucleotides in length, which comprises the nucleotide sequence of SEQ ID NO:1, or the sequence complementary thereto.
In a series of further embodiments, the oligonucleotide can be used in amplification or detection of a nucleic acid sequence found in Vxcex213.1-LGRAGLTY T cells. In one subseries of such embodiments, the oligonucleotide is used in a primer pair, the primer pair comprising or derived from:
(a) a first primer which is an oligonucleotide is from about 15 to 30 nucleotides in length and comprises at least 10 contiguous nucleotides of SEQ ID NO:1, or a sequence complementary thereto; and
(b) a second primer which is an oligonucleotide of about 15 and 30 nucleotides in length that does not comprise the sequence (a), and said second primer sequence can be found in the region from Vxcex2 to Jxcex2 of the Vxcex213.1 gene (SEQ ID NO: 2) in T cell receptor T cells,
wherein the sequences of (a) and (b) are not found on the same strand of the T cell receptor gene.
Preferably said first primer is an oligonucleotide, of about 15 to 30 nucleotides in length, which comprises 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of SEQ ID NO:1, or a sequence complementary thereto. Most preferable is an oligonucleotide, of about 15 to 30 nucleotides in length, which comprises the nucleotide sequence of SEQ ID NO:1, or the sequence complementary thereto.
In another subseries of such embodiments, the oligonucleotide is used as an oligonucleotide probe, the oligonucleotide probe comprising:
(a) an oligonucleotide from about 15 to 30 nucleotide in length and comprises at least 10 contiguous nucleotides of SEQ ID NO:1, or a sequence complementary thereto; and
(b) a labeling moiety.
Preferably, the oligonucleotide, is about 15 to 30 nucleotides in length, and comprises 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of SEQ ID NO:1, or a sequence complementary thereto. Most preferable is an oligonucleotide, of about 15 to 30 nucleotides in length, which comprises the nucleotide sequence of SEQ ID NO:1, or the sequence complementary thereto. The labeling moiety is preferably selected from 32P or digoxingenin.
In another embodiment, the present invention is directed to a method of detecting MBP83-99 Vxcex213.1 T cells expressing a LGRAGLTY motif, comprising:
(i) obtaining a nucleic acid sample from MBP83-99 Vxcex213.1 T cells;
(ii) contacting the nucleic acid sample with a primer pair selected or derived from:
(a) a first primer comprising an oligonucleotide of about 15 to 30 nucleotides in length and comprises at least 10 contiguous nucleotides of SEQ ID NO:1, or a sequence complementary thereto or derived therefrom; and
(b) a second primer comprising and oligonucleotide of about 15 and 30 nucleotides in length that does not comprise the sequence of (a) and is found in the region from Vxcex2 to Jxcex2 of the Vxcex213.1 gene in Vxcex213.1 T cells (SEQ ID NO:2),
wherein the sequences of (a) and (b) are not found on the same strand of the Vxcex213.1 gene; and,
(iii) detecting the presence of the nucleic acid encoding the LGRAGLTY motif.
Preferably the first primer is an oligonucleotide, of about 15 to 30 nucleotides in length, which comprises 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of SEQ ID NO: 1, or a sequence complementary thereto. Most preferable is an oligonucleotide, of about 15 to 30 nucleotides in length, which comprises the nucleotide sequence of SEQ ID NO:1, or the sequence complementary thereto.
Another embodiment of the present invention is directed to peptides, of from 8 to approximately 45 amino acid residues in length, comprising the LGRAGLTY motif (i.e. comprising the sequence of SEQ ID NO: 3). In various aspects of this embodiment, of the invention, the peptide comprises 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 contiguous amino acids of the sequence of SEQ ID NO:32. Preferably the peptide sequence is SEQ ID NO:3 or the sequence is residues 2-21 of SEQ ID NO:32. Most preferably the peptide sequences is residues 2-21 of SEQ ID NO:32.
In yet another embodiment, the present invention is directed to a method of treating an autoimmune disease, comprising:
(a) obtaining MBP83-99 Vxcex213.1 T cells from a human;
(b) detecting the presence of a nucleic acid encoding the LGRAGLTY motif by the method described above; and, if the nucleic acid is detected,
(c) administering one or more peptides of from 8 to approximately 45 amino acid residues in length, each comprising the LGRAGLTY motif, to the human. In various aspects of this embodiment, of the invention, the peptides administered to the human each comprise 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 contiguous amino acids of the sequence of SEQ ID NO:32. Preferably the peptide sequence is SEQ ID NO:3 or the peptide sequence is residues 2-21 of SEQ ID NO:32. Most preferably the peptide sequences is residues 2-21 of SEQ ID NO:32.
In a still further embodiment, the present invention is directed to a method of monitoring an autoimmune disease, comprising:
(a) obtaining MBP83-99 Vxcex213.1 T cells from a human;
(b) detecting the presence of a nucleic acid encoding the LGRAGLTY motif by the method described above; and, if the nucleic acid is detected,
(c) quantifying the nucleic acid.